Gas chromatographic analysis showed acetylcholine to be the only choline ester released from innervated longitudinal muscle of the guinea pigileum by electrical stimulation. The amount of acetylcholine measured by gas chromatography agreed almost exactly with that measured by bioassay. Denervated longitudinal muscle produced no acetylcholine, and treatment of the muscle with tetrodotoxin markedly reduced acetylcholine output. The method permits the recovery and quantitation of amounts of acetylcholine as low as 5 nanograms in 5 milliliters of tissue perfusates.